1. Field of the Invention
This invention relates to methods of assaying pyrrole-containing biological compounds and chemical compositions that can be used in such methods. More specifically, it relates to a method for detecting pyrrole-containing molecules that are markers of particular disease states.
2. Description of Related Art
Ehrlich""s reagent, or p-dimethylaminobenzaldehyde (1), is a molecule that can react with pyrroles and indoles to form a chromogenic compound. 
See G. Lombard and V. Dowell, J. Clin. Microbiol.
(1983) 18:609-613. The mechanism of action is typically described as an electrophilic attack on the xcex1-carbon atom of a pyrrole. This attack forms a highly conjugated cation that absorbs light in the visible spectrum. Such a mechanism is graphically represented in Scheme A above.
The reaction of Ehrlich""s reagent with certain compounds has been discussed. For instance, Iyer reported a pyrrole is formed when LGE2 is reacted with proteins. See Iyer et al., J. Org. Chem. (1994) 59:6038-6043. When the pyrrole was contacted with Ehrlich""s reagent in the presence of BF3.OEt2, a blue-green chromophore was produced. The chromophore was identified as a pyrrolic electrophilic substitution product.
Lombard reported the reaction between Ehrlich""s reagent and bacterially derived indoles. See G. Lombard and V. Dowell, J. Clin. Microbiol. (1983) 18:609-613. The sensitivity of the reagent was compared to two other indole detecting compounds: Kovac""s reagent and DMCA. Ehrlich""s reagent was reported to be 10 times less sensitive than DMCA and 10 times more sensitive than Kovac""s reagent in detecting indole.
While Ehrlich""s reagent has been used to roughly detect the presence of pyrroles or indoles in a targeted material, improved compositions and methods for detecting such heterocycles are desirable, especially methods that provide for detecting pyrrole-containing molecules that are markers of particular disease states.
The present invention provides methods of assaying pyrrole-containing biological compounds. In one case the method involves:
1) contacting the biological compound with either:
a) an optionally labelled derivatizing agent (bound to or able to bind to a solid support), wherein the derivatizing agent forms a reaction product with the biological compound (preferably via covalent attachment thereto), followed by exposure to a detectable molecule which forms a complex with the reaction product; or
b) an optionally labelled derivatizing agent not bound to a solid support, wherein the derivatizing agent forms a reaction product with the biological compound (preferably via covalent attachment thereto), followed by exposure to a binding agent specific to the biological compound in the reaction product, said binding agent being bound to a solid support; or
c) a binding agent bound to a solid support, said binding agent being specific to the biological compound and forming a complex therewith, followed by exposure to an optionally labelled, derivatizing agent which forms a reaction product with the biological compound moiety of said complex (preferably via covalent attachment thereto); and
2) determining the amount of bound biological compound by detecting the detectable molecule, or by determining the amount of free or bond binding agent or by measuring the amount of label present.
Preferably, the method of assaying pyrrole-containing biological compounds is Method 1, described in part a) above. Method 1 involves the following steps:
1) contacting a biological compound with a derivatizing agent of the following structure in the bound form; 
xe2x80x83wherein R1 is an alkyl group, R2 is an alkyl group, A is a linking group and B is a solid support, and wherein the contact induces formation of a reaction product, and wherein the reaction product comprises the covalent attachment of the biological compound to the derivatizing agent; followed by contacting the reaction product with a detectable molecule, wherein the contact induces specific binding of the detectable molecule to the reaction product to provide a complex; and
2) determining the amount of bound biological material by detecting the detectable molecule.
Preferably the detectable molecule is a monoclonal antibody (MAb) specific to the biological compound. Preferably the solid support is a microtitre or a treated glass slide.
Preferably the method of assaying pyrrole-containing biological compounds is Method 2 described in part b) above. Method 2 involves the following steps:
1) contacting the biological compound with an optionally labelled derivatizing agent in solution to form a reaction product therewith (preferably via covalent attachment thereto) followed by exposure to a binding agent bound to a solid support, said binding agent being specific to the biological compound in the reaction product and
2) determining the amount of bound biological compound by determining the amount of labelled derivatizing agent bound to the solid support.
Preferably the derivatizing agent is biotinylated Ehrlich""s reagent. Preferably the solution containing the reaction product is neutralised prior to contact with the bound binding agent. Preferably the bound MAb is bound to a solid support, suitably a microtitre plate or a treated glass slide.
Preferably the derivatizing agent is labelled with a labeling molecule, suitably a radio-labelled, fluorescent label, enzyme label or the like. Preferably the amount of bound biological compound is determined by detecting the amount of labelled derivatizing agent bound on the solid support.
Method 2 takes into account the fact that relatively strong acid conditions are required for the reaction of derivatizing reagents with pyrroles. Thus, most non-covalent interactions, such as antibody-antigen complexes, would be disrupted under these conditions. To overcome this problem, pyrrolic units in the biological sample are targeted in Method 2 by reaction in solution with derivatizing agent to form a reaction product, preferably via covalent attachment thereto followed by capture of the reaction product on a surface coated with specific antibodies.
Preferably, the method of assaying pyrrole-containing biological compounds is Method 3, described in part a) above. Method 3 involves the following steps:
1) contacting a biological compound with a derivatizing agent in solution to form a reaction product wherein the derivatizing agent comprises a first partner of a strong binding pair.
2) contacting the reaction product with a solid support having a second partner of the strong binding pair on its surface, to form a bound complex with the reaction product;
3) contacting the bound complex with a detectable molecule;
4) determining the amount of bound biological compound by detecting the amount of detectable molecule bound to the solid support.
Preferably the derivatizing agent is a p-dimethylaminobenzaldehyde derivative, and in bound form has the following structure: 
wherein R1 is an alkyl group, R2 is an alkyl group, R4 is a heteroalkyl group, X is a first partner of a strong binding pair and Y is a solid support having a second partner of a strong binding pair on its surface.
Preferably the solution containing the reaction product is neutralized prior to contact with the solid support.
In one embodiment the first partner of the strong binding pair is from avidin and the second partner of the strong binding pair is from biotin. Alternatively the first partner of the strong binding pair is from biotin and the second partner of the strong binding pair is from avidin. In a second embodiment the first partner of the strong binding pair is from biotin and the second partner of the strong binding pair is from streptavidin. Alternatively the first partner of the strong binding pair is from streptavidin and the second partner of the strong binding pair is from biotin.
Preferably the detectable molecule is a monoclonal antibody specific to the biological compound moiety of the complex. Suitably the solid support is a microtitre plate or a treated glass slide.
The present invention also provides a method of purifying an antigen, said method comprising;
1) contacting a pyrrole-containing biological compound with one of;
a) an optionally labelled derivatizing agent (bound or able to bind to a solid support) wherein the dirivatizing agent forms a reaction product with the biological compound (preferably via covalent attachment thereto) followed by exposure to a detectable molecule which forms a complex with the reaction product; or
b) an optionally labelled derivatizing agent, not bound to a solid support, wherein the derivatizing agent forms a reaction product with the biological compound (preferably via covalent attachment thereto), followed by exposure to a binding agent bound to a solid support wherein the binding agent is specific to a biological compound in the reaction product; or
c) a binding agent bound to a solid support, said binding agent being specific to the biological compound, and forming a complex therewith, followed by exposure to an optionally labelled, derivatizing agent, which forms a reaction product with the biological compound moiety of said complex (preferably via covalent attachment thereto); and
2) eluting the biological compound from the solid support.
This method allows easy preparation of an antigen, which can then be used in screening for an antigen detection agent, for example antibody.
Preferably the derivatizing agent for use in the method of purifying an antigen is of the following structure in bound form: 
wherein R1 is an alkyl group, R2 is an alkyl group, A is a linking group and B is a solid support.
Preferably the labeled derivatizing agent has the following structure in bound form: 
wherein R1 is an alkyl group, R2 is an alkyl group, R4 is a heteroalkyl group, X is a first partner of a strong binding pair and Y is a solid support having a second partner of a strong binding pair on its surface.
Preferably the detectable molecule is a monoclonal antibody specific to the biological compound.
Optionally the derivatizing agent is labelled with a radio-label, fluorescent label, enzyme label or the like.
The present invention also provides compounds for use in the method of assaying pyrrole-containing biological compounds.
In one case, the compound is of the following structure: 
wherein R1 is an alkyl group, R2 is an alkyl group, A is a linking group and B is a solid support.
More preferably the labeled derivatizing agent has the following structure: 
wherein R1 is a straight-chain alkyl group containing 1 to 10 carbon atoms, R2 is a straight-chain alkyl group containing 1 to 10 carbon atoms, and R4 is a straight-chain heteroalkyl group containing 2 to 10 carbon atoms and at least 2 heteroatoms.